6 research outputs found
New Insights Into Permeation of Large Cations Through ATP-Gated P2X Receptors
The permeability of large cations through the P2X pore has remained arguably the most controversial and complicated topic in P2X-related research, with the emergence of conflicting studies on the existence, mechanism and physiological relevance of a so-called âdilatedâ state. Due to the important role of several âdilatingâ P2X subtypes in numerous diseases, a clear and detailed understanding of this phenomenon represents a research priority. Recent advances, however, have challenged the existence of a progressive, ATP-induced pore dilation, by demonstrating that this phenomenon is an artifact of the method employed. Here, we discuss briefly the history of this controversial and enigmatic dilated state, from its initial discovery to its recent reconsideration. We will discuss the literature in which mechanistic pathways to a large cation-permeable state are proposed, as well as important advances in the methodology employed to study this elusive state. Considering recent literature, we will also open the discussion as to whether an intrinsically dilating P2X pore exists, as well as the physiological relevance of such a large cation-permeable pore and its potential use as therapeutic pathway
Conception and application of new photochemical tools to study P2X receptors
Les rĂ©cepteurs P2X (P2XR), activĂ©s par lâATP, sont impliquĂ©s dans des rĂŽles physiopathologiques. Leur fonctionnement est associĂ© Ă diffĂ©rents Ă©tats conformationnels. Le projet de thĂšse a menĂ© Ă associer la synthĂšse organique et lâapplication de molĂ©cules photo-activables avec des techniques dâĂ©lectrophysiologie patch-clamp, pour dĂ©cortiquer les mouvements molĂ©culaires de ces rĂ©cepteurs et effectuer des relations structure-fonction, via trois stratĂ©gies : - La synthĂšse et application dâagrafes photo-isomĂ©risables qui permet le photo-contrĂŽle des P2XR et lâĂ©tude de mouvements - La synthĂšse et caractĂ©risation dâun acide aminĂ© (aa) photo-clivable pour Ă©tudier les implications de zones sur la fonction des P2XR via une photolyse - Lâincorporation dâun aa non naturel dans les P2XR pour Ă©tudier des interactions et mouvements via un « photo-pontage ». Nous avons Ă©lucidĂ© les mĂ©canismes molĂ©culaires responsables de la permĂ©abilitĂ© des P2XR, rĂ©cusĂ© lâexistence de l'Ă©tat dilatĂ© et identifiĂ© un cation organique physiologique pouvant les traverser. Nous avons aussi conçu un acide aminĂ© photo-clivable pouvant mener Ă des Ă©tudes structure-fonction des P2XR.P2X receptors are cationic ligand-gated ion channels, activated by extracellular ATP, involved in many physio-pathological roles. Their function is associated with different allosteric states. During this PhD, we have designed three new strategies, spanning photochemical organic synthesis and patch-clamp electrophysiology to elucidate the molecular mechanisms involved in these conformational states and to collect data in order to study structure-function relationships. - Synthesis and application of molecular tweezers, which allows the photo-control of P2X Rand the study of molecular motions - Synthesis and characterization of a photo-cleavable amino acid with the aim of incorporating it into P2XR and doing structure-function relationships - Incorporation of an unnatural amino acid for photo-crosslinking studies. We have been able to probe the molecular mechanism involved in large organic cations permeation of P2XR, to bring into question the dilated state and to identify a physiological cation that can flow through P2XR. We have also designed a photo-cleavable amino acid which could serve in the study of structure-function relationships
Conception et application de nouveaux outils photochimiques pour lâĂ©tude des rĂ©cepteurs canaux P2X
P2X receptors are cationic ligand-gated ion channels, activated by extracellular ATP, involved in many physio-pathological roles. Their function is associated with different allosteric states. During this PhD, we have designed three new strategies, spanning photochemical organic synthesis and patch-clamp electrophysiology to elucidate the molecular mechanisms involved in these conformational states and to collect data in order to study structure-function relationships. - Synthesis and application of molecular tweezers, which allows the photo-control of P2X Rand the study of molecular motions - Synthesis and characterization of a photo-cleavable amino acid with the aim of incorporating it into P2XR and doing structure-function relationships - Incorporation of an unnatural amino acid for photo-crosslinking studies. We have been able to probe the molecular mechanism involved in large organic cations permeation of P2XR, to bring into question the dilated state and to identify a physiological cation that can flow through P2XR. We have also designed a photo-cleavable amino acid which could serve in the study of structure-function relationships.Les rĂ©cepteurs P2X (P2XR), activĂ©s par lâATP, sont impliquĂ©s dans des rĂŽles physiopathologiques. Leur fonctionnement est associĂ© Ă diffĂ©rents Ă©tats conformationnels. Le projet de thĂšse a menĂ© Ă associer la synthĂšse organique et lâapplication de molĂ©cules photo-activables avec des techniques dâĂ©lectrophysiologie patch-clamp, pour dĂ©cortiquer les mouvements molĂ©culaires de ces rĂ©cepteurs et effectuer des relations structure-fonction, via trois stratĂ©gies : - La synthĂšse et application dâagrafes photo-isomĂ©risables qui permet le photo-contrĂŽle des P2XR et lâĂ©tude de mouvements - La synthĂšse et caractĂ©risation dâun acide aminĂ© (aa) photo-clivable pour Ă©tudier les implications de zones sur la fonction des P2XR via une photolyse - Lâincorporation dâun aa non naturel dans les P2XR pour Ă©tudier des interactions et mouvements via un « photo-pontage ». Nous avons Ă©lucidĂ© les mĂ©canismes molĂ©culaires responsables de la permĂ©abilitĂ© des P2XR, rĂ©cusĂ© lâexistence de l'Ă©tat dilatĂ© et identifiĂ© un cation organique physiologique pouvant les traverser. Nous avons aussi conçu un acide aminĂ© photo-clivable pouvant mener Ă des Ă©tudes structure-fonction des P2XR
Conception and application of new photochemical tools to study P2X receptors
Les rĂ©cepteurs P2X (P2XR), activĂ©s par lâATP, sont impliquĂ©s dans des rĂŽles physiopathologiques. Leur fonctionnement est associĂ© Ă diffĂ©rents Ă©tats conformationnels. Le projet de thĂšse a menĂ© Ă associer la synthĂšse organique et lâapplication de molĂ©cules photo-activables avec des techniques dâĂ©lectrophysiologie patch-clamp, pour dĂ©cortiquer les mouvements molĂ©culaires de ces rĂ©cepteurs et effectuer des relations structure-fonction, via trois stratĂ©gies : - La synthĂšse et application dâagrafes photo-isomĂ©risables qui permet le photo-contrĂŽle des P2XR et lâĂ©tude de mouvements - La synthĂšse et caractĂ©risation dâun acide aminĂ© (aa) photo-clivable pour Ă©tudier les implications de zones sur la fonction des P2XR via une photolyse - Lâincorporation dâun aa non naturel dans les P2XR pour Ă©tudier des interactions et mouvements via un « photo-pontage ». Nous avons Ă©lucidĂ© les mĂ©canismes molĂ©culaires responsables de la permĂ©abilitĂ© des P2XR, rĂ©cusĂ© lâexistence de l'Ă©tat dilatĂ© et identifiĂ© un cation organique physiologique pouvant les traverser. Nous avons aussi conçu un acide aminĂ© photo-clivable pouvant mener Ă des Ă©tudes structure-fonction des P2XR.P2X receptors are cationic ligand-gated ion channels, activated by extracellular ATP, involved in many physio-pathological roles. Their function is associated with different allosteric states. During this PhD, we have designed three new strategies, spanning photochemical organic synthesis and patch-clamp electrophysiology to elucidate the molecular mechanisms involved in these conformational states and to collect data in order to study structure-function relationships. - Synthesis and application of molecular tweezers, which allows the photo-control of P2X Rand the study of molecular motions - Synthesis and characterization of a photo-cleavable amino acid with the aim of incorporating it into P2XR and doing structure-function relationships - Incorporation of an unnatural amino acid for photo-crosslinking studies. We have been able to probe the molecular mechanism involved in large organic cations permeation of P2XR, to bring into question the dilated state and to identify a physiological cation that can flow through P2XR. We have also designed a photo-cleavable amino acid which could serve in the study of structure-function relationships
Lateral fenestrations in the extracellular domain of the glycine receptor contribute to the main chloride permeation pathway
In-house VMD scripts for counting ion permeation events via the lateral/apical pathways over time and counting the number of chloride ions in the vestibule are provided at the following links: https://doi.org/10.5281/zenodo.6935496 and https://doi.org/10.5281/zenodo.6935478. Molecular snapshots of the zebrafish GlyR-α1 solvated and embedded in a lipid bilayer in the MD-open conformation that were used as starting points for the computational electrophysiology experiments are provided at the following link: https://doi.org/10.5281/zenodo.6529095. All other data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.International audienceGlycine receptors (GlyR) are ligand-gated ion channels mediating signal transduction at chemical synapses. Since the early patch-clamp electrophysiology studies, the details of the ion permeation mechanism have remained elusive. Here, we combine molecular dynamics simulations of a zebrafish GlyR-âș1 model devoid of the intracellular domain with mutagenesis and single-channel electrophysiology of the full-length human GlyR-âș1. We show that lateral fenestrations between subunits in the extracellular domain provide the main translocation pathway for chloride ions to enter/exit a central water-filled vestibule at the entrance of the transmembrane channel. In addition, we provide evidence that these fenestrations are at the origin of current rectification in known anomalous mutants and design de novo two inward-rectifying channels by introducing mutations within them. These results demonstrate the central role of lateral fenestrations on synaptic neurotransmission. Teaser Extracellular chloride ions access the glycine receptor pore via lateral fenestrations outdating the standard apical model
Illumination of a progressive allosteric mechanism mediating the glycine receptor activation
International audiencePentameric ligand-gated ion channel mediate signal transduction at chemical synapses by transiting between resting and open states upon neurotransmitter binding. Here, we investigate the gating mechanism of the glycine receptor fluorescently labeled at the extracellulartransmembrane interface by voltage-clamp fluorimetry (VCF). Fluorescence reports a glycineelicited conformational change that precedes pore opening. Low concentrations of glycine, partial agonists or specific mixtures of glycine and strychnine trigger the full fluorescence signal while weakly activating the channel. Molecular dynamic simulations of a partial agonist boundclosed Cryo-EM structure show a highly dynamic nature: a marked structural flexibility at both the extracellular-transmembrane interface and the orthosteric site, generating docking properties that recapitulate VCF data. This work thus illuminates a progressive propagating transition towards channel opening, displaying structural plasticity with novel implications concerning the mechanism of action of allosteric effectors